Real-time monitoring of recombinant antibody breakthrough during protein A affinity chromatography.

An on-line assay was developed to monitor antibody breakthrough in real time during Protein A affinity chromatography of recombinant antibodies. When loading cell culture fluid on to a Protein A affinity column, antibody breakthrough cannot be measured by UV absorbance because of the flow-through of UV-absorbing impurities. An assay using perfusion chromatography media with immobilized Protein A is a rapid, antibody-specific assay. It directly samples preparative column effluent, allowing real-time measurement of antibody breakthrough during loading of Protein A affinity chromatography. Breakthrough curves were generated for three column media at five flow rates, showing the effects of diffusion on the shape of the breakthrough curve. The breakthrough curves were used to measure dynamic capacity.